Turenne C Y, Sanche S E, Hoban D J, Karlowsky J A, Kabani A M
Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Health Sciences Centre, Winnipeg, Manitoba R3A 1R9, Canada.
J Clin Microbiol. 1999 Jun;37(6):1846-51. doi: 10.1128/JCM.37.6.1846-1851.1999.
Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.
侵袭性真菌病通常在免疫功能低下患者的发病和死亡中起重要作用。当前真菌血培养和组织学检查方法敏感性较差，促使了高敏感性和特异性分子技术的发展，如聚合酶链反应（PCR）。真菌内部转录间隔区2（ITS2）区域的序列变异性可能有助于临床真菌分离株的快速准确诊断。用针对5.8S和28S核糖体DNA（rDNA）保守序列的真菌特异性引物进行PCR，可扩增出物种特异性的ITS2区域，其扩增子长度可变。我们利用ABI PRISM 310基因分析仪和ABI PRISM 310基因扫描分析软件，测定临床上重要真菌（包括念珠菌和非念珠菌酵母、曲霉菌种以及多种皮肤癣菌）ITS2区域的可变大小差异。当样本与人基因组DNA和细菌基因组DNA进行检测时，未发生交叉反应。我们发现，大多数具有临床意义的真菌分离株可用此方法区分，因此它有望成为快速（<7小时）诊断真菌血症和其他侵袭性真菌感染的工具。
